For the preparation of assays in most of the cases several fluid reagents need to be mixed, and for reproducible results of said assays it is of importance that a controlled volume of each reagent and the sample is transferred. Since all fluid transfer processes have a certain variance, transfer errors can not be avoided, but at least it must be possible to detect those transfer processes with unacceptable errors in order to neglect the result of these assays.
Especially real-time PCR systems require an exact mixture of all reaction components for reliable and comparable quantification because the amount of sample material, of primers/probes as well as of master mixes required for real-time PCR particularly influence the quantification result. But of course, other assays also require a set-up with mixing of several components, and a control of said set-up in terms of volume transfer is of importance, too.
To address these uncertainties, normalization techniques can be used, and mainly two different normalization methods are established for quantitative real-time PCR (qPCR):                (1) To normalize against differences of the sample material (DNA or RNA), an unregulated reference gene is measured in combination with the target gene, and the ratio of target/reference is calculated (Relative Quantification method, see, e.g., LightCycler 480 operator's manual, page 179ff).        (2) To normalize against fluorescence intensity differences, a reference dye (not participating in the qPCR reaction) is added, and all measured fluorescence values are normalized against the reference dye fluorescence (Applied Biosystems, ROX reference dye, U.S. Pat. No. 5,736,333).        
But both of these normalization methods exhibit problems. The normalization method (1) can only control the amount/quality of the sample material when relative quantification is applied, not when absolute quantification is used. Furthermore, this method does not control the amount of any other PCR component besides the sample material. The normalization method (2) only normalizes against fluorescence intensity differences generated by qPCR system variations. It does not control or warn the user when the amount of a certain component is below a certain limit.
Currently, there are no fluid transfer control systems known in the state of the art that provide a control system for the volumes of the components to be mixed for an assay set-up.
The present invention provides a closed system for assay set-up that encompasses control strategies to identify volume errors of components mixed for said assays. The method according to the present invention is especially suitable for the set-up of real-time PCR amplifications.